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Adenoviral-induced islet cell cytotoxicity is not counteracted by Bcl-2 overexpression
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2002 (English)In: Molecular medicine (Cambridge, Mass. Print), ISSN 1076-1551, E-ISSN 1528-3658, Vol. 8, no 11, 733-741 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: The ability to transfer immunoregulatory, cytoprotective, or anti-apoptotic genes into pancreatic islet cells may allow enhanced resistance against the autoimmune destruction of these cells in type 1 diabetes. We describe here an inducible transduction system for expression of the anti-apoptotic bcl-2 gene in insulin-producing cells as a potential tool for protecting against beta-cell death.

MATERIALS AND METHODS: Isolated pancreatic rat islet cells or rat insulinoma (RINm5F) cells were transduced using a progesterone antagonist (RU 486) inducible adenoviral vector system, expressing the bcl-2 gene. Bcl-2 overexpression was measured by Western blot assays and flow cytometry analysis. Following exposure to cytokines or to the mitochondrial uncoupler FCCP, cell survival was determined using fluorescence and electron microscopy, and a colorimetric assay (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]- 2H-tetrazolium-5-carboxanilide [XTT]-based) for cell viability. The mitochondrial membrane potential ((m)) was assessed using the lipophilic cationic membrane potential-sensitive dye JC-1.

RESULTS: The adenoviral gene transfer system induced Bcl-2 expression in more than 70% of beta-cells and the protein expression levels were successfully regulated in response to varying concentrations of progesterone antagonist RU 486. Exposure of islet cells to proinflammatory cytokines IL-1beta, TNF-alpha, and IFN-gamma, or to the mitochondrial uncoupler FCCP resulted in disruption of the mitochondrial membrane potential ((m)) and beta-cell death. Bcl-2 overexpression stabilized (m) and prevented cell death in RINm5F cells but not in islet cells. In addition, prolonged in vitro culture revealed adenoviral-induced islet cell necrosis.

CONCLUSIONS: The RU 486-regulated adenoviral system can achieve an efficient control of gene transfer at relatively low doses of the adenoviral vector. However, Bcl-2 overexpression in islet cells did not prevent adenoviral- or cytokine-induced toxicity, suggesting that the specific death pathway involved in adenoviral toxicity in beta-cells may bypass the mitochondrial permeability transition event.

Place, publisher, year, edition, pages
2002. Vol. 8, no 11, 733-741 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-91302PubMedID: 12520090OAI: oai:DiVA.org:uu-91302DiVA: diva2:163988
Available from: 2004-02-03 Created: 2004-02-03 Last updated: 2013-10-01Bibliographically approved
In thesis
1. In vitro Studies of β-cell Death and Survival. Modulation by Adenoviral Vectors and Bcl-2 Overexpression
Open this publication in new window or tab >>In vitro Studies of β-cell Death and Survival. Modulation by Adenoviral Vectors and Bcl-2 Overexpression
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Type 1 diabetes is a multifactorial disease resulting from the selective destruction of insulin-producing β-cells within the pancreatic islets of Langerhans. The mechanisms of β-cell death are not fully understood but cytokines are important mediators of this process. In the present study we found that the combination of IL-1β, TNF-α and IFN-γ induced a nitric oxide-dependent disruption of the mitochondrial membrane potential in rat insulin-producing RINm5F-cells, which seems to be a necessary event for both RINm5F-cell apoptosis and necrosis. The antiapoptotic protein Bcl-2 was able to prevent cellular death in RINm5F cells, most probably by counteracting the mitochondrial permeability transition. These results pointed out the potential of such antiapoptotic genes as gene therapy tools, to allow enhanced resistance against autoimmune destruction of β-cells in type 1 diabetes. For this purpose we used a progesterone-antagonist (RU 486)-inducible gene transfer system to achieve an efficient and controlled Bcl-2 overexpression in primary rat β-cells. However, in our experience, prolonged in vitro culture revealed adenoviral-induced islet cell necrosis, a process that was not prevented by Bcl-2 overexpression. Moreover, we observed that specific adenoviral genotypes correlate with differential induction of necrosis in both human and rat pancreatic islet cells. Although human islet cells showed an increased resistance in terms of viral concentrations required for the induction of cell-toxicity, our results showed that they were unable to build up an efficient antiviral response following infection and that their survival was dependent on the exogenous addition of α-interferon.

In conclusion, adenoviral techniques for overexpression of antiapoptotic proteins in insulin-producing cells may provide useful tools against β-cell directed autoimmune destruction. However, understanding the specific interactions of the viral gene products with cellular proteins and how they are involved in β-cell death regulation is fundamental for an efficient and safe application of gene therapy approaches to type 1 diabetes.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2004. 40 p.
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1320
Cell biology, β-cell, cytokine, mitochondrial membrane potential, Bcl-2, Adenoviral vector, Adenovirus, IFN-α, Diabetes, Cellbiologi
National Category
Cell and Molecular Biology
urn:nbn:se:uu:diva-3973 (URN)91-554-5859-9 (ISBN)
Public defence
2004-02-28, B21, Biomedical Centre, BMC Husargatan 3, Uppsala, 09:15
Available from: 2004-02-03 Created: 2004-02-03Bibliographically approved

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